An in vitro DNA replication system using yeast 2-Mum and ARS plasmid DNA has been developed as a model to investigate the mechanism of DNA replication in eucaryotes. To elucidate the mechanisms of initiation, elongation and termination of DNA replication, this system has been further characterized. Recent biochemical fractionation and reconstitution experiments permitted the purification and characterization of yeast DNA primase, (60,000 daltons), single-stranded DNA binding protein, (38,000 daltons), DNA-dependent ATPase III (63,000 daltons) and DNA polymerase I (140,000 daltons). Furthermore, RNase Hs (21,000, 48,000 and 68,000 daltons) and two other single-stranded DNA binding proteins (14,000 and 20,000 daltons) which might participate in DNA replication have been purified to homogeneity by following their enzymatic activities. To prove that these proteins are required for yeast DNA replication, antibodies have been raised against each and their genes have been identified and cloned from Gammagt11 yeast genomic library using the antibodies. Currently, their nucleotide sequences are being determined. In order to permit identification and isolation of other DNA replication proteins, new temperature-sensitive DNA replication mutants of yeast have been isolated and characterized genetically; some of these mutant genes have been identified and cloned from the yeast genomic library.